The supernatant medium of cultures of peripheral blood leukocytes activated either by phytohemagglutinin (PHA) or dental plaque antigen contains a soluble mediator, osteoclast activating factor (OAF) which stimulates resorption of 19-day fetal rat bones in organ culture; resorption is measured by the release of previously incorporated Ca45 from treated bones relative to release from untreated bones. The effects of OAF in this system has been compared to certain humoral stimulators of resorption. OAF closely resembles parathyroid hormone (PTH) in its biological activity. OAF appears to be chemically different from PTH: OAF is more sensitive to heat-inactivation and more resistant to degradation by certain proteolytic enzymes than PTH; OAF is not retarded by ion exchange resins at neutral pH, unlike PTH; and active OAF supernatants contained very low levels of PTH by immunoassay; these levels were far below the concentrations of PTH required to stimulate resorption in this system. OAF can be distinguished from 1,25-dihydroxycholecalciferol and prostaglandin E2 both by its biological activity and by its chemical characteristics. OAF has been partially purified using Sephadex and ion exchange chromatography. On Sephadex G-100 OAF elutes as a sharp peak between the molecular weight markers of chymotrypsinogen (25,000 daltons) and ribonuclease A (13,700 daltons). Although OAF is not retarded by either DEAE-cellulose or CM- cellulose, chromatography of OAF fraction from Sephadex columns results in a further purification apparently by removal of inhibitory contaminants.